The broad goal of this research program is to understand the link between DNA replication and genome integrity in a model organism, Schizosaccharomyces pombe. The processes of chromosome replication and chromatin assembly are intimately coupled with genome stability and malignancy. Progressive loss of genome integrity can cause or exacerbate cancer. Fission yeast offers a powerful model system with facile genetics, and large chromosomes and replication origins reminiscent of those in metazoan cells. Recent completion of the genome sequence of S. pombe provides new tools allowing rapid identification and characterization of protein function in vivo, making predictions that can then be tested in human cells. The conserved MCM proteins are required both for initiation and elongation stages of DNA synthesis in all eukaryotes. Analysis of MCM function in vivo provides the essential cellular context for their role during S phase. This proposal provides evidence linking MCMs to proteins with multiple roles in DNA metabolism. The first aim will investigate evidence that loss of MCM function leads to double strand breaks in the DNA, and determine how the cell uses proteins of the homologous recombination machinery to prevent this. Interactions between MCMs and recombination machinery will also be examined in human cells. The second aim will characterize MCM function by mutational analysis of residues predicted to play important structural roles, and will characterize the localization of wild type and mutant proteins on replicating chromatin using new methods of visualization. This aim will also analyze modifications of MCM proteins during normal cell cycle and perturbed S phase. The third aim will investigate how chromatin structure is linked to MCM function by examining the links between origin timing and histone modifications, investigating previously identified interactions between histone modifying enzymes and MCMs, and determine how MCMs gain access to DNA within a chromatinized template. All these proteins exist in humans and both have links to cancer, but the in vivo, genetically oriented approach is not possible in tissue culture. However, predictions generated in the yeast system can be tested in human cells. Thus, studies in simple yeast cells have direct relevance to understanding the role of these factors in human cell division. [unreadable] [unreadable]